Poliovirus is a member of the Picornaviridae family of small positive-strand RNA viruses. Diseases associated with these viruses include paralytic poliomyelitis, meningitis, encephalitis, myocarditis, hepatitis and the common cold. Post-polio syndrome is also an important clinical manisfestation that occurrs in patients 30-40 years after the onset of acute poliomyelitis. Other positive-strand RNA viruses that form a large group of important human pathogens include hepatitis C virus, West Nile virus, dengue virus and the SARS-associated coronavirus. During the next grant period, we will continue our studies to define the molecular basis of poliovirus RNA replication using cell-free reactions and reconstituted in vitro assays. Key findings from these experiments will be tested in infected cells and in cells transfected with viral RNA. The primary experimental approach will involve a biochemical analysis of the viral and cellular proteins, viral RNA sequences and protein-RNA interactions that regulate poliovirus replication. HeLa S10 translation- replication reactions and preinitiation RNA replication complexes will be used to characterize the molecular mechanisms that regulate the stability and translation of viral RNA, the uridylylation of VPg and the initiation of (-) and (+) strand RNA synthesis. The primary focus of this work will be to experimentally test a replication model which proposes that the viral genomic RNA forms a circular RNP complex that is required for both for efficient translation and the initiation of (-) strand synthesis. This model also predicts that two different mechanisms are used to initiate (-) and (+) strand RNA synthesis. Uridylylation of VPg on the 3' poly(A) tail is used to initiate (-) strand synthesis. In contrast, VPgpUpU, which is synthesized on the cre(2C) hairpin, is used to prime (+) strand synthesis on the highly conserved sequence at the 3' terminus of (-) strand RNA. Specific aspects of this model will be tested in our ongoing studies that include the following specific aims. (1) Characterize the role of a circular RNP complex in the initiation of (-) strand synthesis. (2) Identify and characterize viral RNA sequences and a cellular host protein that are required for (+) strand initiation. (3) Characterize the proteins and viral RNA sequences that are required for VPgpUpU synthesis. (4) Determine the role of viral and cellular proteins in regulating viral RNA stability and translation.